MOLECULAR COMPONENTS OF ACETYLCHOLINE RECEPTOR CLUSTERS

The early development of nerve-muscle synapses is characterized by three major events: (a) the accumulation of junctional acetylcholine receptors (AChRs), (b) the localization of synaptic acetylcholinesterase (AChE), and (c) the elimination of extrajunctional AChRs. This last event is mechanistically distinct from the accumulation of junctional AChRs and seems to result from the suppression of AChR synthesis in extrajunctional regions of muscle cells caused by muscle contraction. The final result is a muscle cell with a highly elaborated apparatus specialized for efficient synaptic transmission. Experiments in this proposal are designed to understand at the molecular level the way in which neurons and muscle cells communicate to establish this apparatus. The proposal is divided into three major sections. The first will make use of immunocytochemistry and antibody microinjection to demonstrate a functional association between the presence of a newly discovered muscle component (a 37 kilodalton nonmyofibrillar tropomyosin) and the ability to cluster AChRs. This molecule was first identified by its absence from vitally transformed muscle cells which are unable to cluster AChRs at all. The second section of this proposal describes similar techniques to probe other cytoskeletal elements involved in clustering and subsequent structural changes in the muscle cell. These studies are based on the observation from this laboratory that clustering causes a subset of organelles, including myonuclei and the Golgi apparatus, to assume a constant sub-cluster localization. The final section is a study of changes in the levels of AChRs and AChE caused by the increase in muscle cell Ca2+ which occurs during contraction. Also, experiments are designed to test the hypothesis that regional differences in the amount of Ca2+ released during contraction or in the levels of particular Ca2+-binding proteins underly the ability of these cells to specify where particular macromolecules are synthesized. Ca2+ concentration will be measured using the Ca2+-sensitive fluorescent dye fura-2 and optical image processing. Ca2+-binding proteins will be investigated using biochemical and immunological techniques. These experiments should add considerably to our knowledge of how neurons influence properties of their target cells and may contribute to an understanding of a variety of developmental and neurological disorders. In addition, certain results may provide information useful in comprehending cell transformation.